Several procedures are known for treating virus-related diseases and for preventing disorders that arise as a consequence of viral infections. Prophylactic vaccination is probably one of the most effective measures against potential fatal infectious diseases, since an individual can become fully or partly protected against new infections. The immune system makes sure that the virus that has entered the body or cell is prevented from replicating in the individual, and in most cases the virus disappears completely from the system. Therapeutic vaccination refers to the treatment of infected individuals intentionally to prevent the virus from replicating further and consequently to halt disease progression or to cure the disease by eliminating the virus from the body.
Another way of dealing with viral infections is through the use of antiviral agents that either attack the viral particle directly or that prevent the infection, propagation, replication, packaging and/or growth of the virus in the individual. These treatments are applied either when the individual has already been infected and so a prophylactic vaccination is no longer necessary, or when an individual is at immediate risk to encounter an infection. Antiviral molecules inhibit certain processes and phases in the viral life cycle, thereby inhibiting the virus from spreading.
Several in vitro and in vivo methods for the identification of such antiviral compounds are known in the art. Methods that make use of the specific antiviral activity of certain compounds include the plaque reduction assay, the yield reduction assay, the virus antigen determination assay, the dye-uptake assay, the cytopathic effect (CPE) determination assay and several in vivo assays for virus replication. Many of the in vitro methods, especially the plaque reduction assay, have the major disadvantage that they cannot be applied in (very) high-throughput screens. Although the plaque reduction assay can be applied for most viruses that are known to date, it is necessary to inoculate large numbers of susceptible cells in suitable conditions with ranges of virus titers as well as large ranges of antiviral compound titers to detect the correct concentration of the compound that significantly decreases the number of plaques. This situation makes the plaque reduction assay very suitable for measuring the right concentration of a specific compound that affects the growth of a particular virus but very unsuitable for the identification of such (new and unknown) compounds in a library. Since many of the molecule libraries consist of a very large collection of separate compounds (>1014 individual agents), it is required to have settings in which all separate compounds can be screened in a rapid and efficient manner with low costs. The other in vitro methods such as the yield reduction assay, the virus antigen determination assay, the dye-uptake assay and the cytopathic effect (CPE) determination assay are to a certain extent more suitable for high-throughput screening, but they clearly depend on the cell line that is used and whether such a cell line is able to grow in multi-well settings and for prolonged periods of time. Clearly, many of the primary cells that are used to determine the effect of an antiviral compound in plaque reduction assays cannot be cultured in high-throughput settings, since these cells do not grow indefinitely. Evidently, the in vivo antiviral methods, such as for example the ferret-, the mouse- and chicken models for influenza infection (reviewed by Sidwell et al. 2000) are useless for the identification of novel compounds that prevent virus-cell recognition and virus infection, replication, propagation and growth, especially when high-throughput settings are preferred.
Many susceptible non-continuous cells have been identified in which most viruses propagate. As mentioned, these cells can be used in assays such as the plaque reduction assay but cannot be applied for screening of antiviral compounds, since they do not either grow in multi-well formats or they do not grow indefinitely. Only a limited number of continuous cell lines have been identified that support the growth of certain viruses. These cell lines include the green monkey VERO cells, the Madin-Darby Canine Kidney (“MDCK”) cells, the human lung embryo MRC-5 cells and the human A549 cells. However, a major drawback of these cells is that they only support the growth of a limited number of viruses, while not all of these cell lines are capable of continuous growth in multi-well formats. Nevertheless, a number of drugs displayed antiviral activities against viruses such as CMV, Influenza and HSV in the context of using the cells mentioned above. For example, Acyclovir, an approved purine nucleoside analogue, inhibited HSV replication in A549 cells (Li et al. 1988). Despite the few successful propagations of certain viruses on continuous cell lines and the prevention of propagation by a number of antiviral compounds, it was found that in many cases the cells did not support the complete life cycle of the mentioned viruses. This limits their use significantly in screening assays for antiviral compounds present in large libraries, because the life cycle of a virus is built up from several phases in which a compound can have its point of impact.
Although many cell-based systems exist that can be used to determine whether a particular compound is capable of preventing certain phases in the life cycle of a virus, no system is believed to be present in the art that combines the possibility of screening large numbers of (possible) antiviral compounds in a very high-throughput setting with the possibility of screening a large range of different viruses. No system is available in the art that combines these possibilities to determine the antiviral activity of a certain compound present in a compound library, in different phases of the life cycle of the particular virus that is attacked by this particular compound.